Hepatitis C Viruses (HCV) constitute a senus within the Flaviviridae, with closest homology to the hepatitis G and GB viruses, and Pestiviruses. The positive-stranded RNA genome encodes at least 9 proteins. Core, E1, and E2 constitute the structural proteins. NS2, NS3, NS4A, NS4B, NS5A, and NS5B are non-structural (NS) proteins. HCV isolates display high levels of sequence heterogeneity allowing classification into at least 11 types and 90 subtypes (Maertens and Stuyver, 1997). HCV infection of the human liver is often clinically benign, with mild icterus in the acute phase. The disease may even go unnoticed in some cases of acute resolving hepatitis C. In the majority (>70%) of cases, however, HCV infection leads to chronic persistent or active infection, often with complications of liver cirrhosis and auto-immune disorders. Hepatocellular carcinoma may occur after about 20 to 35 years (Saito et al., 1990), sometimes even without the intermediate phase of cirrhosis. No prophylaxis is available today and treatment with interferon-alpha (IFN-α) only leads to long-term resolution in about 4 to 36% of treated cases, depending on the HCV genotype (Maertens and Stuyver, 1997).
Since productive culture methods for HCV are currently not available, and since only minute amounts of HCV antigens circulate in the infected patient, direct detection of HCV particles cannot be performed routinely, and indirect diagnosis is only possible using cumbersome amplification techniques for HCV RNA detection. Unlike with many other viral infections, HCV particles generally persist in the blood, liver, and lymphocytes despite the presence of cellular and humoral immune response to most of the HCV proteins. HCV antibodies can be conveniently detected by Elisa techniques which allow high throughput screening in blood banks and clinical laboratories. Supplementary antibody testing is required and is now mandatory in most countries. True HCV reactivity is thus discriminated from false reactivity, which may be caused by non-specific binding of serum or plasma immunoglobulines or anti-idiotypic components to the coating or blocking reagents, or to contaminants present in HCV antigen preparations, or even to fusion parts or non-specific regions of the recombinant antigens themselves (McFarlane et al., 1990). HCV RNA detection by PCR or branched DNA (bDNA) techniques have recently been introduced to monitor chronic HCV disease, especially during therapy. Surprisingly, HCV RNA detection is sometimes employed to confirm HCV Ab screening tests, despite the fact that only ˜70-94% of repeatedly HCV Ab positive patient samples are positive by nested PCR (Marin et al., 1994). Of HCV Ab positive blood donors, who usually present with milder forms of the disease and low HCV RNA levels, confirmation by nested PCR is usually in the order of ˜40% (Waumans et al., 1993; Stuyver et al., 1996). Strip-based assays therefore provide the only reliable alternative for HCV Ab confirmation. Even in the case of an indeterminate result in the confirmatory assay, serological follow up of the patient rather than HCV RNA detection is advisable (Di Biscealie et al., 1998). Since native HCV antigens are not available insufficient quantities, such confirmatory assays incorporate synthetic peptides and/or recombinant fragments of HCV proteins. One of the most critical issues in the confirmation of antibodies constitutes the reactivity of the NS3 protein (Zaaijer et al., 1994). NS3 antibodies often appear first in seroconversion series and the reactivity of the NS3 protein seems to be different in the different commercial assays available today.
Innogenetics introduced the concept of strip technology in which usually a combination of synthetic peptides and recombinant proteins are applied as discrete lines in an ordered and easily readable fashion. The INNO-LIA HIV Ab tests have proven to be superior to routinely used western blots (Pollet et al., 1990). The Line Immuno Assay allows multiparameter testing and thus enables incorporation of cutoff and other rating systems, sample addition control, as well as testing for false reactivity to non-HCV proteins used as carrier or fusion partner required for some antigens in the Elisa test. In principle, the test format allows to combine antigens of different aetiological agents or phenotypically linked conditions into a single test.
The INNO-LIA HCV Ab III is a 3rd generation Line Immuno Assay which incorporates HCV antigens derived from the Core region, the E2 hypervariable region (HVR), the NS3 helicase region, and the NS4A, NS4B, and NS5A regions. In the third generation assay, highly purified recombinant subtype 1b NS3 protein and E2 peptides enabled superior sensitivity while safeguarding the reliable specificity which is characteristic of peptide-based tests (Peeters et al., 1993). Perhaps one of the most important features of this assay is its unprecedented correlation with HCV RNA positivity (Claeys et al., 1992; De Beenhouwer et al., 1992).
The antigens are coated as 6 discrete lines on a nylon strip with plastic backing. In addition, four control lines are coated on each strip: anti-streptavidin, 3+positive control (anti-human Ig), 1+ positive control (human IgG), and the ± cutoff line (human IgG). A diluted test sample is incubated in a trough together with the LIA III strip. If present in the sample, HCV antibodies will bind to the HCV antigen lines on the strip. Subsequently, an affinity-purified alkaline phosphatase labelled goat anti-human IgG (H+L) conjugate is added and reacts with specific HCV antigen/antibody complexes if previously formed. Incubation with enzyme substrate produces a chestnut-like color, the intensity of which is proportionate to the amount of HCV-specific antibody captured from the sample on any given line. Color development is stopped with sulphuric acid. If no HCV-specific antibodies are present, the conjugate only binds to the ±, 1+, and 3+ control lines. If the addition of sample is omitted, only the ± and 1+ control lines will be stained.